Optical diffraction biosensor

ABSTRACT

The present invention provides an inexpensive and sensitive device and method for detecting and quantifying analytes present in a medium. The device comprises a metalized film upon which is printed a specific, predetermined pattern of analyte-specific receptors. Upon attachment of a target analyte to select areas of the plastic film upon which the receptor is printed, diffraction of transmitted and/or reflected light occurs via the physical dimensions and defined, precise placement of the analyte. A diffraction image is produced which can be easily seen with the eye or, optionally, with a sensing device.

TECHNICAL FIELD

The present invention is in the field of analyte sensors and, morespecifically the present invention is in the field of microcontactprinting binders on metal films to produce optical diffractionbiosensors.

BACKGROUND OF THE INVENTION

Microcontact printing is a technique for forming patterns of organicmonolayers with micron and submicron lateral dimensions. It offersexperimental simplicity and flexibility in forming certain types ofpatterns. In the prior art, microcontact printing was used withself-assembled monolayers of long-chain alkanethiolates to form organicstructures on gold and other metals. These patterns acted as nanometerresists by protecting the supporting metal from corrosion byappropriately formulated etchants, or, allowed for the selectiveplacement of fluids on hydrophilic regions of the pattern. In general,patterns of self-assembled monolayers having dimensions that can be lessthan 1 micron are formed by using the alkanethiol as an "ink", and byprinting them on the metal support using an elastomeric "stamp". Thestamp is fabricated by molding a silicone elastomer using a masterprepared by optical or X-ray microlithography or by other techniques.(See U.S. patent application Ser. Nos. 08/654,993; 08/769,594;08/821,464; 08/707,456 and 08/768,449 which are incorporated herein intheir entirety by reference)

Microcontact printing brings to microfabrication a number of newcapabilities. Microcontact printing makes it possible to form patternsthat are distinguished only by their constituent functional groups; thiscapability permits the control of surface properties such as interfacialfree energies with great precision. In the prior art microcontactprinting relies on molecular self-assembly. Using self-assemblingmonolayers, a system is generated that is (at least locally) close to athermodynamic minimum and is intrinsically defect-rejecting andself-healing. Simple procedures, with minimal protection against surfacecontamination by adsorbed materials or by particles, can lead tosurprisingly low levels of defects in the final structures. Theprocedure using self-assembling monolayers can be conducted atatmospheric pressure, in an unprotected laboratory atmosphere. Thus,microcontact printing that uses self-assembling monolayers is useful inlaboratories that do not have routine access to the equipment normallyused in microfabrication, or for which the capital cost of equipment isa serious concern. The patterned self-assembled monolayers can bedesigned to act as resists with a number of wet-chemical etchants.

Also in the prior art, a gold film 5 to 2000 nanometers thick istypically supported on a titanium-primed Si/SiO₂ wafer or glass sheet.The titanium serves as an adhesion promoter between gold and thesupport. However, the silicon wafer is rigid, brittle, and cannottransmit light. These silicon wafers are also not suitable for alarge-scale, continuous printing process, such as in letterpress,gravure, offset, and screen printing (see Printing Fundamentals, A.Glassman, Ed. (Tappi Press Atlanta, Ga. 1981); Encyclopedia Britannica,vol. 26, pp. 76-92, 110-111 (Encyclopedia Brittanica, Inc. 1991)). Inaddition, silicon must be treated in a separate step with an adhesionpromoter such as Cr or Ti, or Au will not adequately adhere, preventingformation of a stable and well-ordered monolayer. Finally, silicon isopaque to visible light, so any diffraction pattern obtained must becreated with reflected, not transmitted light.

What is needed is an easy, efficient and simple method of contactprinting a patterned receptor on an optically transparent, flexiblesubstrate, that is amenable to continuous processing and does not useself-assembling monolayers. Such a method and the device resulting fromsuch a method is simpler, not restricted to the limitations ofself-assembling monolayers and is easier to manufacture.

SUMMARY OF THE INVENTION

The present invention provides an inexpensive and sensitive device andmethod for detecting and quantifying analytes present in a medium. Thedevice comprises a metalized film upon which is printed a specificpredetermined pattern of analyte-specific receptor. The presentinvention does not utilize self-assembling monolayers but is moregeneral in that any receptor which can be chemically coupled to asurface can be used. Upon attachment of a target analyte which iscapable of scattering light to select areas of the plastic film uponwhich the receptor is printed, diffraction of transmitted and/orreflected light occurs via the physical dimensions, refractive index anddefined, precise placement of the analyte. In the case where an analytedoes not scatter visible light because the analyte is too small or doesnot have an appreciable refactive index difference compared to thesurrounding medium, the attachment of polymer beads coupled with theanalyte to receptors is another method of producing diffraction oflight. A diffraction image is produced which can be easily seen with theeye or, optionally, with a sensing device. The present invention is abiosensor comprising a polymer film coated with metal and a receptorlayer printed onto the polymer film wherein the receptor layer has areceptive material thereon that specifically binds an analyte.

The present invention utilizes methods of contact printing of patternedmonolayers utilizing derivatives of binders for microorganisms. Oneexample of such a derivative is a thiol. The desired binders can bethiolated antibodies or antibody fragments, proteins, nucleic acids,sugars, carbohydrates, or any other functionality capable of binding ananalyte. The derivatives are chemisorbed to metal surfaces such asmetalized polymer films.

Patterned monolayers allow for the controlled placement of analytesthereon via the patterns of analyte-specific receptors. The biosensingdevices of the present invention produced thereby are used by firstexposing the biosensing device to a medium that contains the analyte ofchoice and then, after an appropriate incubation period, transmittinglight, such as from a laser or a point light source, through the film.If the analyte is present in the medium and is bound to the receptors onthe patterned monolayer, the light is diffracted in such a way as toproduce a visible or near infrared image. In other words, the patternedmonolayers with the analyte bound thereto can produce opticaldiffraction patterns which differ depending on the reaction of thereceptors on the monolayer with the analyte of interest. The light canbe in the visible spectrum, and be either reflected from the film, ortransmitted through it, and the analyte can be any compound or particlereacting with the monolayer. The light can be a white light ormonochromatic electromagnetic radiation in preferably the visibleregion. The present invention also provides a flexible support for amonolayer on gold or other suitable metal or metal alloy.

The present invention includes a support for a thin layer of gold orother suitable material which does not require an adhesion promoter forthe formation of a well-ordered monolayer or thin layer of binder. Thepresent invention also provides a support for a layer of gold or othermaterial which is suitable for continuous printing, rather than batch,fabrication. In addition, the present invention provides a low-cost,disposable biosensor which can be mass produced. The biosensors of thepresent invention can be produced as a single test for detecting ananalyte or it can be formatted as a multiple test device. The uses forthe biosensors of the present invention include, but are not limited to,detection of chemical or biological contamination in garments, such asdiapers, generally the detection of contamination by microorganisms inprepacked foods such as fruit juices or other beverages and the use ofthe biosensors of the present invention in health diagnosticapplications such as diagnostic kits for the detection of antigens,microorganisms, and blood constituents.

In another embodiment of the present invention, nutrients for a specificclass of microorganisms can be incorporated into the receptor monolayer.In this way, very low concentrations of microorganisms can be detectedby first contacting the biosensor of the present invention with thenutrients incorporated therein and then incubating the biosensor underconditions appropriate for the growth of the bound microorganism. Themicroorganism is allowed to grow until there are enough organisms toform a diffraction pattern.

The present invention can also be used on contact lenses, eyeglasses,window panes, pharmaceutical vials, solvent containers, water bottles,bandaids, and the like to detect contamination.

These and other features and advantages of the present invention willbecome apparent after a review of the following detailed description ofthe disclosed embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a metal plated MYLAR film with anutrient backing.

FIG. 2 shows a biosensor capable of simultaneously measuring severaldifferent analytes in a medium.

FIG. 3 is a schematic of contact printing of receptors according to thepresent invention.

FIG. 4 is an enzyme-linked immunosorbent assay (ELISA) of the surfaceprinted with a thiolated antibody binder.

FIG. 5 is a photograph showing polystyrene surrogate particles coatedwith antigen after attachment to the printed antibody.

FIG. 6 is a diffraction pattern produced from the sample described inFIG. 4.

FIG. 7 is an optical photomicrograph of Candida albicans attached to apatterned antibody receptor.

FIG. 8 is a diffraction pattern caused by the binding of Candidaalbicans to the patterned receptor.

DETAILED DESCRIPTION

The present invention features improved biosensing devices, and methodsfor using such biosensing devices, for detecting and quantifying thepresence or amount of an analyte of interest within a medium. Theanalytes that can be detected by the present invention include, but arenot limited to, microorganisms such as bacteria, yeasts, fungi andviruses. In contrast to prior devices, those of the present inventionallow detection of extremely small quantities of analyte in a medium ina rapid assay lasting only a few minutes. In addition, other than alight source, no signaling or associated electronic components arerequired in the biosensing devices of the present invention.

The present invention comprises micro-contact printing ofanalyte-specific receptors (thiolated binders) onto metalized plasticfilm which allows for the development of single use, disposablebiosensors based on light diffraction to indicate the presence of theanalyte. Upon attachment of a target analyte to select areas of theplastic film which contain the receptor, diffraction of transmittedand/or reflected light occurs via the physical dimensions and defined,precise placement of the analyte. For example, yeast, fungi or bacteriumare large enough to act as diffraction elements for visible light whenplaced in organized patterns on a surface.

In addition to producing a simple diffraction image, patterns ofanalytes can be such as to allow for the development of a holographicsensing image and/or a change in visible color. Thus, the appearance ofa hologram or a change in an existing hologram will indicate a positiveresponse. The pattern made by the diffraction of the transmitted lightcan be any shape including, but not limited to, the transformation of apattern from one pattern to another upon binding of the analyte to thereceptive material. In particularly preferred embodiments, thediffraction pattern becomes discernible in less than one hour aftercontact of the analyte with the biosensing device of the presentinvention.

The diffraction grating which produces the diffraction of light uponinteraction with the analyte must have a minimum periodicity of about1/2 the wavelength and real or imaginary a refractive index differentfrom that of the surrounding medium. Very small analytes, such asviruses or molecules, can be detected indirectly by using a largerparticle that is specific for the small analyte. One embodiment in whichthe small analyte can be detected comprises coating the particle, suchas a latex bead or polystyrene bead, with a receptive material, such asan antibody, that specifically binds to the analyte of interest.Particles that can be used in the present invention include, but are notlimited to, glass, cellulose, synthetic polymers or plastics, latex,polystyrene, polycarbonate, proteins, bacterial or fungal cells and thelike. The particles are desirably spherical in shape, but the structuraland spatial configuration of the particles is not critical to thepresent invention. For instance, the particles could be slivers,ellipsoids, cubes, random shape and the like. A desirable particle sizeranges from a diameter of approximately 0.2 μm to 50 μm, desirablybetween approximately 0.4 μm to 1 μm. The composition of the particle isnot critical to the present invention.

It is to be understood that the optimal particle size is a function ofthe refractive index of the particle and the refractive index of thesurrounding medium. A method of analyzing the optimal particle size foruse in the present invention with a transmission image is by employingthe equation;

    t.sub.opt =λ/2(n.sub.2 -n.sub.1)

wherein

t_(opt) =optimum height of the particle

λ=wavelength of incoming light

n₂ =refractive index of particle

n₁ =refractive index of surrounding medium

For a reflection image, the optimum hight of the particle is determinedby the above equation divided by two.

The monolayer on the metalized film contains a receptive material orbinder, such as an antibody, that will specifically bind to an epitopeon the analyte that is different from the epitope used in the binding tothe particle. Thus, for detecting a medium with a small analyte, such asviral particles, the medium is first exposed to the latex particles towhich the viral particles are bound. Then, the latex particles areoptionally washed and exposed to the metalized film with the monolayerscontaining the virus specific antibodies. The antibodies then bind tothe viral particles on the latex bead thereby immobilizing the latexbeads in the same pattern as the monolayers on the film. Because thebound latex beads will cause diffraction of the visible light, adiffraction pattern is formed, indicating the presence of the viralparticle in the liquid. Other combinations using particles are wellknown to those of ordinary skill in the art.

In another embodiment of the present invention, receptors, such asantibodies are attached to the metal layer as described herein. Theantibodies are then exposed to an environment that contains analytesthat bind to the receptor. After the analyte has bound to the receptor,a second receptor is added that recognizes the metal bound conjugate. Tothis second receptor is bound an enzyme or inorganic substance that willcause a precipitation of a solid substance when the appropriate reagentor reagents are added. For example, an enzyme that can cause aprecipitate to form is peroxidase in the presence oftetramethylbenzidene (See Example 3 herein). Another example, is the useof colloidal gold in the presence of a silver halide. Elemental silverwill precipitate on the patterned receptor layer thereby producing thediffraction pattern.

The analytes that are contemplated as being detected using the presentinvention include, but are not limited to, bacteria; yeasts; fungi;viruses; rheumatoid factor; antibodies, including, but not limited toIgG, IgM, IgA and IgE antibodies; carcinoembryonic antigen;streptococcus Group A antigen; viral antigens; antigens associated withautoimmune disease, allergens, tumor antigens; streptococcus Group Bantigen, HIV I or HIV II antigen; or host response (antibodies) to theseand other viruses; antigens specific to RSV or host response(antibodies) to the virus; antigen; enzyme; hormone; polysaccharide;protein; lipid; carbohydrate; drug or nucleic acid; Salmonella species;Candida species, including, but not limited to Candida albicans andCandida tropicalis; Neisseria meningitides groups A, B, C, Y and W sub135, Streptococcus pneumoniae, E. coli, Haemophilus influenza type B; anantigen derived from microorganisms; a hapten; a drug of abuse; atherapeutic drug; an environmental agent; and antigens specific toHepatitis.

In another embodiment of the present invention, nutrients for a specificclass of microorganism can be incorporated into the monolayer. In thisway, very low concentrations of microorganisms can be detected by firstcontacting the biosensor of the present invention with the nutrientsincorporated therein and then incubating the biosensor under conditionsappropriate for the growth of the bound microorganism. In one embodimentshown in FIG. 1, the MYLAR film 15 has a nutrient backing 30 that is incontact with the back of the MYLAR film 15. The opposite side of theMYLAR film 15 has a metal film 20 thereon. The metal film 20 ispreferably gold. Attached to the metal film 20 are receptors 25 that arespecific for a microorganism. In use, the nutrient diffuses slowlythrough the MYLAR film. When a microorganism is attached to receptor 25,the bound microorganism consumes the nutrient and grows. As themicroorganism grows, it diffracts impinging light thereby forming adiffraction pattern. Thus, in this embodiment, if the diffractionpattern forms, it is because the bound microorganism grew. Of course, insome cases, the microorganism can multiply enough to form a diffractionpattern without the presence of a nutrient on the patterned monolayer.

A part of the present invention is a receptive material that can bemicroprinted on the metalized film and will specifically bind to theanalyte of interest. Thus, the receptive material is defined as one partof a specific binding pair and includes, but is not limited to,antigen/antibody, enzyme/substrate, oligonucleotide/DNA, chelator/metal,enzyme/inhibitor, bacteria/receptor, virus/receptor, hormone/receptor,DNA/RNA, or RNA/RNA, oligonucleotide/RNA, and binding of these speciesto any other species, as well as the interaction of these species withinorganic species.

The receptive material that is bound to the attachment layer ischaracterized by an ability to specifically bind the analyte or analytesof interest. The variety of materials that can be used as receptivematerial is limited only by the types of material which will combineselectively (with respect to any chosen sample) with a secondarypartner. Subclasses of materials which fall in the overall class ofreceptive materials include toxins, antibodies, antibody fragments,antigens, hormone receptors, parasites, cells, haptens, metabolites.allergens, nucleic acids, nuclear materials, autoantibodies, bloodproteins, cellular debris, enzymes, tissue proteins. enzyme substrates,coenzymes. neuron transmitters, viruses, viral particles,microorganisms, proteins, polysaccharides, chelators, drugs, and anyother member of a specific binding pair. This list only incorporatessome of the many different materials that can be coated onto theattachment layer to produce a thin film assay system. Whatever theselected analyte of interest is, the receptive material is designed tobind specifically with the analyte of interest.

The matrix containing the analyte of interest may be a liquid, a solid,or a gas, and can include a bodily fluid such as mucous, saliva, urine,fecal material, tissue, marrow, cerebral spinal fluid, serum, plasma,whole blood, sputum, buffered solutions, extracted solutions, semen,vaginal secretions, pericardial, gastric, peritoneal, pleural, or otherwashes and the like. The analyte of interest may be an antigen, anantibody, an enzyme, a DNA fragment, an intact gene, a RNA fragment, asmall molecule, a metal, a toxin, an environmental agent, a nucleicacid, a cytoplasm component, pili or flagella component, protein,polysaccharide, drug, or any other material, such as those listed inTable A. For example, receptive material for bacteria may specificallybind a surface membrane component, protein or lipid, a polysaccharide, anucleic acid, or an enzyme. The analyte which is specific to thebacteria may be a polysaccharide, an enzyme, a nucleic acid, a membranecomponent, or an antibody produced by the host in response to thebacteria. The presence of the analyte may indicate an infectious disease(bacterial or viral), cancer or other metabolic disorder or condition.The presence of the analyte may be an indication of food poisoning orother toxic exposure. The analyte may indicate drug abuse or may monitorlevels of therapeutic agents.

One of the most commonly encountered assay protocols for which thistechnology can be utilized is an immunoassay. However, the generalconsiderations apply to nucleic acid probes, enzyme/substrate, and otherligand/receptor assay formats. For immunoassays, an antibody may serveas the receptive material or it may be the analyte of interest. Thereceptive material, for example an antibody or an antigen, must form astable, dense, reactive layer on the attachment layer of the testdevice. If an antigen is to be detected and an antibody is the receptivematerial, the antibody must be specific to the antigen of interest; andthe antibody (receptive material) must bind the antigen (analyte) withsufficient avidity that the antigen is retained at the test surface. Insome cases, the analyte may not simply bind the receptive material, butmay cause a detectable modification of the receptive material to occur.This interaction could cause an increase in mass at the test surface ora decrease in the amount of receptive material on the test surface. Anexample of the latter is the interaction of a degradative enzyme ormaterial with a specific, immobilized substrate. In this case, one wouldsee a diffraction pattern before interaction with the analyte ofinterest, but the diffraction pattern would disappear if the analytewere present. The specific mechanism through which binding,hybridization, or interaction of the analyte with the receptive materialoccurs is not important to this invention, but may impact the reactionconditions used in the final assay protocol.

In general, the receptive material may be passively adhered to theattachment layer in a pattern that will produce a diffraction pattern.If required, the free functional groups introduced onto the test surfaceby the attachment layer may be used for covalent attachment of receptivematerial to the test surface. Chemistries available for attachment ofreceptive materials are well known to those skilled in the art.

A wide range of techniques can be used to adhere the receptive materialto the attachment layer in a pattern that, when bound to the analyte ofinterest, forms a diffraction pattern when light is transmitted throughattachment layer. Test surfaces may be coated with receptive material byapplication of solution in discrete arrays or patterns; spraying, inkjet, or other imprinting methods; or by spin coating from an appropriatesolvent system. The technique selected should minimize the amount ofreceptive material required for coating a large number of test surfacesand maintain the stability/functionality of receptive material duringapplication. The technique must also apply or adhere the receptivematerial to the attachment layer in a very uniform and reproduciblefashion.

The receptor layer is formed from material selected from the groupconsisting of antigens, antibodies, oligonucleotides, chelators,enzymes, bacteria, bacterial pili, bacterial flagellar materials,nucleic acids, polysaccharides, lipids, proteins, carbohydrates, metals,viruses, hormones and receptors for said materials In the preferredembodiments, the biosensing device is configured and arranged to providea pattern detectable by eye in response to transmission of polychromaticlight when the analyte of interest is sandwiched between the receptivematerial and a secondary binding reagent.

The medium in which the analyte may reside can be solid, gel-like,liquid or gas. For purposes of detecting an analyte in a body fluid, thefluid is selected from the group consisting of urine, serum, plasma,spinal fluid, sputum, whole blood, saliva, uro-genital secretions, fecalextracts, pericardial, gastric, peritoneal, pleural washes, vaginalsecretions, and a throat swab; and the method optionally includes usinga diffractometer to measure the appearance of the diffraction pattern.The most common gas that is contemplated as being used with thebiosensing device of the present invention is air.

The biosensing device of the present invention utilizes methods ofcontact printing of patterned monolayers on metalized polymer films,desirably thermoplastic polymer films, the compositions producedthereby, and the use of these compositions. Patterned monolayers allowfor the controlled placement of fluids thereon which can contain aanalyte receptor. The term "patterned monolayers thereon" as used hereinmeans the monolayers in any pattern on the metalized polymer filmsincluding a solid pattern.

When the film with the monolayers thereon is exposed to an analyte thatis capable of reacting with the monolayer, the film will produce opticaldiffraction patterns which differ depending on the reaction of themonolayer with the analyte of interest. The liquid may be a high surfacetension fluid such as water. The light can be in the visible spectrum,and be either reflected from the film, or transmitted through it, andthe analyte can be any compound reacting with the monolayer.

In preferred embodiments, the method involves contacting the substratewith a test sample potentially containing the analyte under conditionsin which the substrate causes a change in the refractive index of themonolayer. When light is transmitted through the metalized thermoplasticpolymer with the monolayer, a visible pattern is formed and can bevisualized by directing the light to a surface or by looking directlythrough the substrate.

In one embodiment, the present invention is contemplated in a dipstickform in which the micro-contact printed metalized film is mounted at theend of the dipstick. In use the dipstick is dipped into the liquid inwhich the suspected analyte may be present and allowed to remain forseveral minutes. The dipstick is then removed and then, either a lightis projected through the metalized film or the film is observed with alight behind the film. If a pattern is observed, then the analyte ispresent in the liquid.

In another embodiment of the present invention, a multiple analyte testis constructed on the same support. As shown in FIG. 2, a strip 50 isprovided with several micro-contact printed metalized films 70, 75, 80and 85, each film having a monolayer pattern 60 printed thereon. Each ofthe micro-contact printed metalized films 70, 75, 80 and 85 have adifferent receptive material that is different for different analytes.It can be seen that the present invention can be formatted in any arraywith a variety of micro-contact printed metalized films thereby allowingthe user of the biosensor device of the present invention to detect thepresence of multiple analytes in a medium using a single test.

In yet another embodiment of the present invention, the biosensor can beattached to an adhesively backed sticker or decal which can then beplaced on a hard surface or container wall. The biosensor can be placedon the inside surface of a container such as a food package or a glassvial. The biosensor can then be visualized to determine whether there ismicrobial contamination.

In one embodiment of the present invention, the receptor layer has thefollowing general formula:

    X--R--Y

X is reactive with metal or metal oxide. For example, X may beasymmetrical or symmetrical disulfide (--R'SSR, --RSSR), sulfide(--R'SR, --RSR), diselenide (--R'Se--SeR), selenide (--R'SeR, --RSeR),thiol (--SH), nitrile (--CN), isonitrile, nitro (--NO₂), selenol(--SeH), trivalent phosphorous compounds, isothiocyanate, xanthate,thiocarbamate, phosphine, thioacid or dithioacid, carboxylic acids,hydroxylic acids, and hydroxamic acids.

R is an linker which may optionally be interrupted by hetero atoms andwhich are preferably non-branched for the sake of optimum dense packing.The linker, helps prevent steric hindrance and/or enhance activity of Y.

Y is the molecule that imparts functionality of the receptor layer. Ycan be any molecule that preferentially binds the analyte of interest. Ycan be toxins, antibodies, antibody fragments antigens, hormonereceptors, parasites, cells, haptens, metabolizes. allergens, nucleicacids, nuclear materials, autoantibodies, blood proteins, cellulardebris, enzymes, tissue proteins. enzyme substrates, coenzymes. neurontransmitters, viruses, viral particles, microorganisms, proteins,polysaccharides, chelators, drugs, and any other member of a specificbinding pair.

A desired reagent that can be reacted with potential binders such asantibodies or antibody fragments to provide functionality X, include,but are not limited to, sulfo-LC-SPDP (Pierce Chemical Co. Rockford,Ill.) which has the following formula: ##STR1##

The sulfo-LC-SPDP is reactive towards sulfhydryl and amino groups and istherefore ideally suited for reaction with proteins such as antibodiesor other protein receptors, proteoglycans, lipoproteins, glycoproteins,or amino sugars including, but not limited to, glucosamine orgalactosamine.

In a typical experimental procedure, schematically shown in FIG. 3, aphotolithographically produced master is placed in a glass or plasticPetri dish, and a 10:1 ratio (w:w or v:v) mixture or SYLGARD® siliconeelastomer 184 and SYLGARD® silicone elastomer 184 curing agent (DowCorning Corporation) is poured over it. The elastomer is allowed to sitfor approximately 30 minutes at room temperature and reduced pressure todegas, then cured for 4 to 16 hours at 60° C., and gently peeled fromthe master.

"Inking " of the elastomeric stamp is accomplished by soaking theelastomeric stamp in an approximately 0.1 mG/mL to approximately 0.5mG/mL concentration of the receptor "ink" for between approximately 10seconds to 10 minutes, followed by drying the stamp under nitrogen gas.The stamp is allowed to dry until no liquid is visible by eye on thesurface of the stamp (typically about 60 seconds), either under ambientconditions, or by exposure to a stream of nitrogen gas. Followinginking, the stamp is applied (typically by hand) to a metalized surface.Very light hand pressure is used to aid in complete contact between thestamp and the surface. The stamp should desirably remain on the surfacefor between approximately 10 seconds to approximately 200 seconds. Theactual time the stamp should remain on the surface will vary dependingupon the ink used. The stamp is then gently peeled from the surface. Apreferred embodiment will follow receptor printing with a passivationstep to cover all the surface area of the metal not containing boundreceptor. Passivation helps eleminate non-specfic binding of analyte.

The elastomeric character of the stamp is important to the success ofthe process. Polydimethylsiloxane (PDMS), when cured, is sufficientlyelastomeric to allow good conformal contact of the stamp and thesurface, even for surfaces with significant relief; this contact isessential for efficient contact transfer of the receptor "ink" to thegold film. The elastomeric properties of PDMS are also important whenthe stamp is removed from the master: if the stamp were rigid (as is themaster) it would be difficult to separate the stamp and master aftercuring without damaging one of the two substrates. PDMS is alsosufficiently rigid to retain its shape, even for features withsub-micron dimensions. Patterns have been successfully generated withlines as small as 200 nm in width. The surface of PDMS has a lowinterfacial free energy (y=22.1 dynes/cm), and the stamp does not adherestrongly to the metalized film. The stamp is durable in that the samestamp can be used up to 100 times over a period of several monthswithout significant degradation in performance. The polymeric nature ofPDMS also plays a critical role in the inking procedure, by enabling thestamp to absorb the alkanethiol ink without significant swelling. Thestamp can be produced on a printing roll to allow for a continuousprinting operation.

A more detailed description of the methods and compositions of thepresent invention follows. All publications cited herein areincorporated by reference in their entirety.

Any thermoplastic film upon which a metal substrate can be deposited issuitable for the present invention. These include, but are not limitedto, polymers such as: polyethylene-terephthalate (MYLAR®),acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylatecopolymer, cellophane, cellulosic polymers such as ethyl cellulose,cellulose acetate, cellulose acetate butyrate, cellulose propionate,cellulose triacetate, cellulose triacetate, polyethylene,polyethylene--vinyl acetate copolymers, ionomers (ethylene polymers)polyethylene-nylon copolymers, polypropylene, methyl pentene polymers,polyvinyl fluoride, and aromatic polysulfones. Preferably, the plasticfilm has an optical transparency of greater than 80%. Other suitablethermoplastics and suppliers may be found, for example, in referenceworks such as the Modern Plastics Encyclopedia (McGraw-Hill PublishingCo., New York 1923-1996).

In one embodiment of the invention, the thermoplastic film with themetal coating thereon has an optical transparency of betweenapproximately 5% and 95%. A more desired optical transparency for thethermoplastic film used in the present invention is betweenapproximately 20% and 80%. In a desired embodiment of the presentinvention, the thermoplastic film has at least an approximately 80%optical transparency, and the thickness of the metal coating is such asto maintain an optical transparency greater than about 20%, so thatdiffraction patterns can be produced by either reflected or transmittedlight. This corresponds to a metal coating thickness of about 20 nm.However, in other embodiments of the invention, the gold thickness maybe between approximately 1 nm and 1000 nm.

The preferred metal for deposition on the film is gold. However, silver,aluminum, chromium, copper, iron, zirconium, platinum and nickel, aswell as oxides of these metals, may be used. Chromium oxide can be usedto make metalized layers.

In principle, any surface with corrugations of appropriate size could beused as masters. The process of microcontact printing starts with anappropriate relief structure, from which an elastomeric stamp is cast.This `master` template may be generated photolithographically, or byother procedures, such as commercially available diffraction gratings.In one embodiment, the stamp may be made from polydimethylsiloxane.

In one embodiment, the present invention features an optical assaydevice, having an optically active receptive surface configured andarranged to allow simultaneous assay of a plurality of samples on thesurface for one analyte of interest, and an automated liquid handlingapparatus (e.g., a pipetting device) configured and arranged to dispensesample and reagent solutions to the surface.

The present invention has a broad range of applications and, may beutilized in a variety of specific binding pair assay methods. Forexample, the devices of this invention can be used in immunoassaymethods for either antigen or antibody detection. The devices may beadapted for use in direct, indirect, or competitive detection schemes,for determination of enzymatic activity, and for detection of smallorganic molecules (e.g., drugs of abuse, therapeutic drugs,environmental agents), as well as detection of nucleic acids.

The stamp may be applied in air, or under a fluid such as water toprevent excess diffusion of the alkanethiol. For large-scale orcontinuous printing processes, it is most desirable to print in air,because shorter contact times are desirable for those processes.

In one embodiment of the present invention, the pattern is formed on themetalized thermoplastic polymer with the receptor layer. In anotherembodiment of the present invention, the relief of the pattern is formedwith the receptor layer. After the stamping process, the metalized areason the plastic may optionally be passivated, for example, with amethyl-terminated monolayer such as hexadecylmercaptan.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention.

EXAMPLE 1

An example derivatization of an antibody to E. coli 0157:H7 (Kirkegaard& Perry Labs) follows: To 1 mG of antibody was added 450 μL phosphatebuffered saline (PBS) and 50 μL of a 10 mM aqueous solution ofSulfo-LC-SPDP (Pierce Catalog #21650). After 60 minutes at roomtemperature, the solution is desalted in a D-Salt™ Polyacrylamidedesalting column (Pierce, Rockford, Ill.) An acetate buffer, pH 4.5 wasused if a subsequent reduction of the disulfide bond was done, while aPBS buffer, pH 7.5, was used if the antibody derivative was to remain asthe disulfide. 500 μL fractions were collected from the column, and thefraction with antibody derivative was determined using a Coomassie® PlusProtein Assay.

EXAMPLE 2

The resulting thiolated antibody from Example 1, either disulfide orthiol, is contact printed on gold-coated MYLAR®. The elastomeric stampis soaked in a 0.5 mG/mL concentration of the thiolated antibody for 10minutes, followed by drying the stamp under nitrogen gas, and thencontacted with a gold-coated MYLAR® film for 10-120 seconds.

EXAMPLE 3

In this example, a condensation figure is produced. The non-patternedareas after printing as described in Example 2 are reacted with anotherthiol, such as hexadecanethiol. The condensation figure remained,indicating that the thiolated antibody is chemisorbed and not displaced.An enzyme-linked immunosorbent assay (ELISA) of the printed surface waspositive in the patterned areas, verifying the presence of activeantibody in the pattern (FIG. 4). The ELISA utilized a peroxidaseconjugated with an antibody specific for the E. coli antibody used inExample 1. Tetramethylbenzidene precipitation on the patterned antibodysandwich produced the diffraction pattern. Polystyrene surrogateparticles surface modified with antigen also produced patternedadsorption to the receptor layer. The diffraction pattern produced bythe tetramethylbenzidene precipitation is shown in FIG. 6.

EXAMPLE 4

A surrogate polystyrene particle was produced by carbodiimide couplingwith ethyldimethylaminodicarbodiimide (EDAC-Sigma Chemical Company, St.Louis, Mo.) of E. coli 0157:H7 antigen(Kirkegaard & Perry Labs, Cat#50-95-90) to one micron polystyrene latex spheres by conventionaltechniques. A 2 wt % solution of the diimide was reacted withcarboxylate modified PS latex(Bangs: 10¹⁰ particles/mL) for 4 hours,followed by exposing these activated particles to a 400 uG/mL solutionof antigen. This surrogate, diluted to 10⁸ particles/mL, was exposed toa sensor containing patterned antibody to E. Coli 0157:H7, produced asdescribed in Example 2, for 60 minutes. After washing with phosphatebuffer, the sample was dried, photographed (FIG. 5) and was shown toproduce a diffraction pattern as described in Example 3.

EXAMPLE 5

It is well established that one criteria for the presence of a selfassembling monolayer (SAM) is increased resistance to chemical etchantsand that alkane thiol self assembled monolayers provide resistance ofgold to cyanide etching. Cyanide etching was performed to determine ifthe thiolated protein or oligonucleotide binders of this invention forma protective SAM on gold. Gold coated polyester film (35 nM goldthickness) was exposed to aqueous solutions of either a thiolatedantibody to Candida albicans (0.5 mG/mL), thiolated protein G (0.5mG/mL), thiolated oligonucleotide (10 μM), or underivatized antibody toCandida albicans (physisorption only; 0.5 mG/mL); an ethanol solution ofhexadecane thiol (HDT; 5.7 mM), known to form a SAM on gold, was used asa positive control. After 16 hours exposure to the thiol containingbinders, the coated gold samples were removed, thoroughly rinsed withsolvent (water or ethanol), and dried under a nitrogen stream.

Binder coated samples were immersed in an aqueous solution of potassiumcyanide (100 mM) containing potassium hydroxide (0.5 M) while bubblingair (oxygen) into the solution. After etching for 11 minutes, thesamples were removed, rinsed with water, and visually evaluated for theamount of gold remaining. Table 1 summarizes the observations. The HDTsample was the only sample with most of the gold remaining on itssurface. The thiolated antibody had a very small amount of gold (≈5%coverage), while the other samples had no gold remaining after etching.This demonstrates that unlike HDT, the thiolated binders used to preparethe optical diffraction biosensors do not form a protective SAM.

                  TABLE 1                                                         ______________________________________                                        Summary of Cyanide Etching Experiments                                                                 Observations                                           Sample after etching                                                        ______________________________________                                        Hexadecane thiol (HDT)                                                                             70-80%                                                     Thiolated Antibody ˜5% (random specks)                                  Antibody (physisorbed on surface) no gold remaining                           Thiolated Protein G no gold remaining                                         Thiolated Oligonucleotide no gold remaining                                 ______________________________________                                    

EXAMPLE 6

Samples with patterned antibody to Candida albicans were prepared asfollows: Gold/polyester(10 nM gold thickness) was pre-treated byimmersing it in a 5 mG/mL phosphate-buffered saline solution (pH 7.2) ofbeta casein (Sigma catalog # C6905) for 10 minutes. The sample wasthoroughly rinsed with distilled water, and dried under a strongnitrogen stream. Contact printing was done using a polydimethyl siloxanestamp having an x,y array of 10-micron diameter circles. The stamp wascoated with a thiolated antibody to Candida albicans (initial polyclonalantibody was Catalog # 20-CRO4 from Fitzgerald Industries International,Inc., Concord, Mass.) by immersing the stamp in a 0.5 mG/mL aqueoussolution of the antibody derivative. After 10 minutes, the stamp wasremoved and thoroughly dried using a strong stream of nitrogen. Contactprinting was done on the casein-treated sample, with exposure times of 1second to 2 minutes being adequate. Two minutes was the preferredcontact time. After printing, the sample was again rinsed with distilledwater and dried.

The sensor sample was exposed to germ tube-bearing cells of Candidaalbicans by inoculating tape-stripped adult forearm skin with aconcentration of 10⁶ yeast cells per milliliter, and placing the sensoron top of the yeast containing tape. Transfer of the yeast cells to thesensor was accomplished after only a few seconds of contact (FIG. 7).Patterned adhesion of the yeast cells to the sensor was confirmed bymicroscopic analysis, and resulted in a diffraction image uponirradiation with a laser (FIG. 8).

Other surfaces which have been inoculated with germ tube-bearing cellsof Candida albicans have been an agar plate and a HUGGIES® Baby Wipe.Exposure of these surfaces to the antibody-based sensors has alsoresulted in patterned attachment of the cells, and diffraction images.

Those skilled in the art will now see that certain modifications can bemade to the invention herein disclosed with respect to the illustratedembodiments, without departing from the spirit of the instant invention.And while the invention has been described above with respect to thepreferred embodiments, it will be understood that the invention isadapted to numerous rearrangements, modifications, and alterations, allsuch arrangements, modifications, and alterations are intended to bewithin the scope of the appended claims.

We claim:
 1. A biosensor comprising:a polymer film coated with metal; and a patterned receptor layer printed onto the polymer film wherein the receptor layer has a receptive material thereon that specifically binds an analyte;wherein the receptor layer is printed in a pattern such that when the biosensor binds an analyte, the biosensor diffracts transmitted light or reflected light to form a diffraction pattern visible to an unaided eye.
 2. The biosensor of claim 1, wherein the diffraction pattern forms a hologram.
 3. The biosensor of claim 1, wherein the metal is gold, silver, chromium, nickel, platinum, aluminum, iron, copper, chromium oxide or zirconium.
 4. The biosensor of claim 3, wherein the metal is gold.
 5. The biosensor of claim 3, wherein the metal coating is between approximately 1 nanometer and 1000 nanometers in thickness.
 6. The biosensor of claim 1, wherein the polymer film is polyethylene-terephthalate, acrylonitrile-butadiene-styrene, acrylonitrile-methyl acetate copolymer, cellophane, ethyl cellulose, cellulose acetate, cellulose acetate butyrate, cellulose propionate, cellulose triacetate, polyethylene, polyethylene-vinyl acetate copolymers, ionomers (ethylene polymers) polyethylene-nylon copolymers, polypropylene, methyl pentene polymers, polyvinyl fluoride, or aromatic polysulfones.
 7. The biosensor of claim 6, wherein the polymer film is polyethylene-terephthalate.
 8. The biosensor of claim 1, wherein the polymer film is optically transparent.
 9. The biosensor of claim 1, wherein the polymer film has an optical transparency between 5% and 95%.
 10. The biosensor of claim 1, wherein the polymer film has an optical transparency between approximately 20% and 80%.
 11. The biosensor of claim 1, wherein the patterned receptor layer is formed from compounds with the following general formula:

    X--R--Y

wherein: X is reactive with the metal or metal oxide on the polymer film; R is an optional linker; and Y is a compound with any property of interest.
 12. The biosensor of claim 11, wherein R is between 0 and 12 carbon atoms in length.
 13. The biosensor of claim 11, wherein X is generated from a compound comprising the following formula: ##STR2##
 14. The biosensor of claim 1, wherein the analyte is bacteria, yeast, fungus, virus, rheumatoid factor, IgG, IgM, IgA and IgE antibodies, carcinoembryonic antigen, streptococcus Group A antigen, viral antigens, antigens associated with autoimmune disease, allergens, tumor antigens, streptococcus Group B antigen, HIV I or HIV II antigen, antibodies viruses, antigens specific to RSV, an antibody, antigen, enzyme, hormone, polysaccharide, protein, lipid, carbohydrate, drug, nucleic acid, Neisseria meningitides groups A, B, C, Y and W sub 135, Streptococcus pneumoniae, E. coli K1, Haemophilus influenza type B, an antigen derived from microorganisms, a hapten, a drug of abuse, a therapeutic drug, an environmental agents, or antigens specific to Hepatitis.
 15. The biosensor of claim 14, wherein the analyte is bacteria, yeast, fungus or virus.
 16. The biosensor of claim 1, wherein the receptor material is antigens, antibodies, nucleotides, chelators, enzymes, bacteria, yeasts, fungi, viruses, bacterial pili, bacterial flagellar materials, nucleic acids, polysaccharides, lipids, proteins, carbohydrates, metals, hormones and receptors for said materials.
 17. The biosensor of claim 16, wherein the fungus is Candida species.
 18. The biosensor of claim 16, wherein the bacteria is Salmonella species.
 19. The biosensor of claim 1, wherein the biosensor is attached to the inside wall of a container.
 20. The biosensor of claim 19, wherein the container is a vial.
 21. The biosensor of claim 18, wherein the container is a food container.
 22. The biosensor of claim 1, wherein the biosensor is attached to the inside wall of a garment.
 23. The biosensor of claim 21, wherein the garment is a diaper.
 24. The biosensor of claim 1, wherein the analyte is attached to a particle.
 25. The biosensor of claim 24, wherein the particle is comprised of glass, cellulose, latex, polystyrene, polycarbonate, protein, or microbial cells.
 26. The biosensor of claim 24, wherein the particle is between approximately 0.2 nm and 50 nm.
 27. The biosensor of claim 26, wherein the particle is between approximately 0.4 μm to 1 μm.
 28. The biosensor of claim 26, wherein the particle size is determined by the following formula:

    t.sub.opt =λ/2(n.sub.2 -n.sub.1)

wherein t_(opt) =optimum height of the particle λ=wavelength of incoming light n₂ =refractive index of particle n₁ =refractive index of surrounding medium.
 29. A method of making a biosensor comprising applying a receptor layer to a polymer film coated with metal in a pattern such that when an analyte binds to the patterned receptor layer, the patterned receptor layer thereby forms a diffraction image visible to an unaided eye. 